AP180 and AP-2 interact directly in a complex that cooperatively assembles clathrin
AP180 and AP-2 interact directly in a complex that cooperatively assembles clathrin
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DOI：
10.1074/jbc.274.32.22785
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发表时间：
1999-08-06
影响因子：
4.8
通讯作者：
Lafer, EM
中科院分区：
文献类型：
--
作者：
Hao, WH;Luo, Z;Lafer, EM
网格蛋白包被的囊泡参与真核细胞细胞内区室之间的蛋白质和脂质运输，AP-2和AP180是网格蛋白包被的囊泡在神经末梢的常驻外壳蛋白，这些蛋白质之间的相互作用在囊泡动力学中可能很重要，AP180和AP-2各自在酸性条件下有效地组装网格蛋白，但两种蛋白质都不会在生理pH下有效地组装网格蛋白。我们发现 AP180 和 AP-2 之间存在直接的、不依赖于网格蛋白的相互作用，并且 AP180-AP-2 复合物在生理条件下组装网格蛋白比单独的任一蛋白质更有效。 AP180 在体内被磷酸化，并且在粗囊泡提取物中，其磷酸化通过酪蛋白激酶 II 的刺激而增强，已知酪蛋白激酶 II 存在于包被的囊泡中。我们发现重组 AP180 是体外酪蛋白激酶 II 的底物，其磷酸化削弱了 AP180 与 AP-2 的结合以及这些蛋白质的协同网格蛋白组装活性。我们将AP-2的结合位点定位于AP180的氨基酸623-680，AP180/AP-2相互作用可以被含有AP-2结合位点的重组AP180片段破坏，并且该片段还破坏AP180-AP-2复合物的协同网格蛋白组装活性。这些结果表明AP180和AP-2直接相互作用形成复合物，比单独的任一蛋白质更有效地组装网格蛋白。通过调节 AP180 对 AP-2 的亲和力，AP180 的磷酸化可能有助于体内网格蛋白组装的调节。
Clathrin-coated vesicles are involved in protein and lipid trafficking between intracellular compartments in eukaryotic cells, AP-2 and AP180 are the resident coat proteins of clathrin-coated vesicles in nerve terminals, and interactions between these proteins could be important in vesicle dynamics, AP180 and AP-2 each assemble clathrin efficiently under acidic conditions, but neither protein will assemble clathrin efficiently at physiological pH. We find that there is a direct, clathrin-independent interaction between AP180 and AP-2 and that the AP180-AP-2 complex is more efficient at assembling clathrin under physiological conditions than is either protein alone. AP180 is phosphorylated in vivo, and in crude vesicle extracts its phosphorylation is enhanced by stimulation of casein kinase II, which is known to be present in coated vesicles. We find that recombinant AP180 is a substrate for casein kinase II in vitro and that its phosphorylation weakens both the binding of AP-2 by AP180 and the cooperative clathrin assembly activity of these proteins. We have localized the binding site for AP-2 to amino acids 623-680 of AP180, The AP180/AP-2 interaction can be disrupted by a recombinant AP180 fragment containing the AP-2 binding site, and this fragment also disrupts the cooperative clathrin assembly activity of the AP180-AP-2 complex, These results indicate that AP180 and AP-2 interact directly to form a complex that assembles clathrin more efficiently than either protein alone. Phosphorylation of AP180, by modulating the affinity of AP180 for AP-2, may contribute to the regulation of clathrin assembly in vivo.